Methods for inhibiting vascular smooth muscle cell proliferation and restinosis

ABSTRACT

Methods of inhibiting vascular smooth muscle cell proliferation and vascular restinosis comprising administering to a human or other mammal in need of treatment an effective amount of a compound having the formula ##STR1## wherein R 1  and R 3  are independently hydrogen, ##STR2## wherein Ar is optionally substituted phenyl; R 2  is ##STR3## R 4  is hydrogen or --OR 1 , and pharmaceutically acceptable salts and solvates thereof.

This application is a division, of application Ser. No. 08/138,296,filed Oct. 15, 1993 now U.S. Pat. No. 5,457,113.

BACKGROUND OF THE INVENTION

Smooth muscle cell proliferation plays an important role in diseasessuch as atherosclerosis and restenosis. Vascular restenosis afterpercutaneous transluminal coronary angioplasty (PTCA) has been shown tobe a tissue response characterized by an early and late phase. The earlyphase occuring hours to days after PTCA is due to thrombosis with somevasospasms while the late phase appears to be dominated by excessiveproliferation and migration of smooth muscle cells. In this disease, theincreased cell motility and colonization by smooth muscle cells andmacrophages contribute significantly to the pathogenesis of the disease.The excessive proliferation and migration of vascular smooth musclecells may be the primary mechanism to the reocclusion of coronaryarteries following PTCA, atherectomy, laser angioplasty and arterialbypass graft surgery. See "Intimal Proliferation of Smooth Muscle Cellsas an Explanation for Recurrent Coronary Artery Stenosis afterPercutaneous Transluminal Coronary Angioplasty," Austin et al., Journalof the American College of Cardiology 8:369-375 (Aug. 1985).

Vascular restenosis remains a major long term complication followingsurgical intervention of blocked arteries by percutaneous transluminalcoronary angioplasty (PTCA), atherectomy, laser angioplasty and arterialbypass graft surgery. In about 35% of the patients who undergo PTCA,reocclusion occurs within three to six months after the procedure. Thecurrent strategies for treating vascular restenosis include mechanicalintervention by devices such as stents or pharmacologic therapiesincluding heparin, low molecular weight heparin, coumarin, aspirin, fishoils calcium antagonists steroids, and prostacyclin. These strategieshave failed to curb the reocclusion rate and have been ineffective forthe treatment and prevention of vascular restenosis. See "Prevention ofRestenosis after Percutaneous Transluminal Coronary Angioplasty: TheSearch for a `Magic Bullet`," Hermans et al., American Heart Journal122:171-187 (July 1991).

In the pathogenesis of restinosis excessive cell proliferation andmigration occurs as a result of growth factors produced by cellularconstituents in the blood and the damaged arterial vessel wall whichmediate the proliferation of smooth muscle cells in vascular restenosis.

Agents that inhibit the proliferation and/or migration of smooth musclecells are useful in the treatment and prevention of restenosis. Thepresent invention provides for the use of compounds as smooth musclecell proliferation inhibitors.

SUMMARY OF THE INVENTION

The invention provides a method of inhibiting smooth muscle cellproliferaton in a human or other mammal subject comprising administeringto said subject a pharmaceutically effective dose of a compound of theformula ##STR4## wherein R₁ and R₃ are independently hydrogen, ##STR5##wherein Ar is optionally substituted phenyl; R₂ ##STR6## R₄ is hydrogenor --OR₁, and pharmaceutically acceptable salts and solvates thereof.Also provided is a method for inhibiting restinosis.

DETAILED DESCRIPTION OF THE INVENTION

The current invention concerns the discovery that a select group ofcompounds, those of formula I and II, are useful for inhibiting smoothmuscle cell proliferation and restinosis. The methods of treatmentprovided by this invention are practiced by administering to a human orother mammal in need a dose of a compound of formula I or II, or apharmaceutically acceptable salt or solvate thereof, that is effectiveto inhibit smooth muscle cell proliferation or restinosis. The terminhibit is defined to include its generally accepted meaning whichincludes phrophylactically treating a human subject to incurring smoothmuscle cell proliferaton or restinosis, and holding in check and/ortreating existing smooth muscle cell prolifertion or restinosis. Assuch, the present method includes both medical therapeutic and/orprophylactic treatment, as appropriate.

Generally, the compound is formulated with common excipients, diluentsor carriers, and compressed into tablets, or formulated as elixirs orsolutions for convenient oral administration, or administered by theintramuscular or intravenous routes. The compounds can be administeredtransdermally, and may be formulated as sustained release dosage formsand the like.

The compounds of formula I used in the methods of the current inventioncan be made according to established procedures, such as those detailedin U.S. Pat. Nos. 4,133,814, 4,418,068, and 4,380,635 all of which areincorporated by reference herein. In general, the process starts with abenzo[b]thiophene having a 6-hydroxyl group and a 2-(4-hydroxyphenyl)group. The starting compound is protected, alkylated, and deprotected toform the formula I compounds. Examples of the preparation of suchcompounds are provided in the U.S. patents discussed above. Thecompounds of formula II may be prepared as described in U.S. Pat. Nos.4,230,862 and 4,232,707, incorporated herein by reference.

Included in the invention is the use of the following compounds:##STR7##

Substituted phenyl includes phenyl substituted once or twice with C₁ -C₆alkyl, C₁ -C₄ alkoxy, hydroxy, nitro, chloro, fluoro, or tr(chloro orfluoro)methyl.

The compounds used in the methods of this invention formpharmaceutically acceptable acid and base addition salts with a widevariety of organic and inorganic acids and bases and include thephysiologically acceptable salts which are often used in pharmaceuticalchemistry. Such salts are also part of this invention. Typical inorganicacids used to form such salts include hydrochloric, hydrobromic,hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric and the like.Salts derived from organic acids, such as aliphatic mono anddicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoicand hydroxyalkandioic acids, aromatic acids, aliphatic and aromaticsulfonic acids, may also be used. Such pharmaceutically acceptable saltsthus include acetate, phenylacetate, trifluoroacetate, acrylate,ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate,methoxybenzoate, methylbenzoate, o-acetoxybenzoate,naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate,γ-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, caprate,caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate,heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate,malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate,oxalate, phthalate, teraphthalate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate,propionate, phenylpropionate, salicylate, sebacate, succinate, suberate,sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,benzene-sulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate,ethanesulfonate, 2-hydroxyethanesulfonate, methane-sulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate,xylenesulfonate, tartarate, and the like. A preferable salt is thehydrochloride salt.

The pharmaceutically acceptable acid addition salts are typically formedby reacting a compound of formula I with an equimolar or excess amountof acid. The reactants are generally combined in a mutual solvent suchas diethyl ether or benzene. The salt normally precipitates out ofsolution within about one hour to 10 days and can be isolated byfiltration or the solvent can be stripped off by conventional means.

Bases commonly used for formation of salts include ammonium hydroxideand alkali and alkaline earth metal hydroxides, carbonates andbicarbonates, as well as aliphatic and aromatic amines, aliphaticdiamines and hydroxy alkylamines. Bases especially useful in thepreparation of addition salts include ammonium hydroxide, potassiumcarbonate, sodium bicarbonate, calcium hydroxide, methylamine,diethylamine, ethylene diamine, cyclohexylamine and ethanolamine.

The pharmaceutically acceptable salts generally have enhanced solubilitycharacteristics compared to the compound from which they are derived,and thus are often more amenable to formulation as liquids or emulsions.

Pharmaceutical formulations can be prepared by procedures known in theart. For example, the compounds can be formulated with commonexcipients, diluents, or carriers, and formed into tablets, capsules,suspensions, powders, and the like. Examples of excipients, diluents,and carriers that are suitable for such formulations include thefollowing: fillers and extenders such as starch, sugars, mannitol, andsilicic derivatives; binding agents such as carboxymethyl cellulose andother cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; moisturizing agents such as glycerol; disintegrating agentssuch as agaragar, calcium carbonate, and sodium bicarbonate; agents forretarding dissolution such as paraffin; resorption accelerators such asquaternary ammonium compounds; surface active agents such as cetylalcohol, glycerol monostearate; adsorptive carriers such as kaolin andbentonire; and lubricants such as talc, calcium and magnesium stearate,and solid polyethyl glycols.

The compounds can also be formulated as elixirs or solutions forconvenient oral administration or as solutions appropriate forparenteral administration, for instance by intramuscular, subcutaneousor intravenous routes. Additionally, the compounds are well suited toformulation as sustained release dosage forms and the like. Theformulations can be so constituted that they release the activeingredient only or preferably in a particular part of the intestinaltract, possibly over a period of time. The coatings, envelopes, andprotective matrices may be made, for example, from polymeric substancesor waxes.

The particular dosage of a compound of formula I required to inhibitsmooth muscle cell proliferation and restenosis according to thisinvention will depend upon the severity of the condition, the route ofadministration, and related factors that will be decided by theattending physician. Generally, accepted and effective daily doses willbe from about 0.1 to about 1000 mg/day, and more typically from about 50to about 200 mg/day. Such dosages will be administered to a subject inneed of treatment from once to about three times each day, or more oftenas needed to effectively inhibit smooth muscle cell proliferation orrestinosis.

The local delivery of inhibitory amounts of active compound for thetreatment of restinosis can be by a variety of techniques whichadminister the compound at or near the proliferative site. Examples oflocal delivery techniques are not intended to be limiting but to beillustrative of the techniques available. Examples include localdelivery catheters, site specific carriers, implants, direct injection,or direct applications.

Local delivery by a catheter allows the administration of apharmaceutical agent directly to the proliferative lesion. Examples oflocal delivery using a balloon catheter are described in EPO 383 492 A2and U.S. Pat. No. 4,636,195 (Wolinsky, Jan. 13, 1987).

Local delivery by an implant describes the surgical placement of amatrix that contains the pharmaceutical agent into the proliferativelesion. The implanted matrix releases the pharmaceutical agent bydiffusion, chemical reaction, or solvent activators. Lange, Science249:1527-1533 (September, 1990).

An example of local delivery by an implant is the use of a stent. Stentsare designed to mechanically prevent the collapse and reocclusion of thecoronary arteries. Incorporating a pharmaceutical agent into the stentdelivers the drug directly to the proliferative site. Local delivery bythis technique is described in Kohn, Pharmaceutical Technology (Octobers1990).

Another example is a delivery system in which a polymer that containsthe pharmaceutical agent is injected into the lesion in liquid form. Thepolymer then cures to form the implant in situ. This technique isdescribed in PCT WO 90/03768 (Donn, Apr. 19, 1990).

Another example is the delivery of a pharmaceutical agent by polymericendoluminal sealing. This technique uses a catheter to apply a polymericimplant to the interior surface of the lumen. The pharmaceutical agentincorporated into the biodegradable polymer implant is thereby releasedat the surgical site. It is described in PCT WO 90/01969 (Schindler,Aug. 23, 1989).

A final example of local delivery by an implant is by direct injectionof vesicles or microparticulates into the proliferative site. Thesemicroparticulates may be composed of substances such as proteins,lipids, carbohydrates or synthetic polymers. These microparticulateshave the pharmaceutical agent incorporated throughout the microparticleor over the microparticle as a coating. Delivery systems incorporatingmicroparticulates are described in Lange, Science 249: 1527-1533(September, 1990) and Mathiowitz, et al., J. App. Poly. Sci., 26:809(1981).

Local delivery by site specific carriers describes attaching thepharmaceutical agent to a carrier which will direct the drug to theproliferative lesion. Examples of this delivery technique includes theuse of carriers such as a protein ligand or a monoclonal antibody.Lange, Science 249:1527-1533 (September).

Local delivery by direct application includes the use of topicalapplications. An example of a local delivery by direct application isapplying the pharmaceutical agent directly to the arterial bypass graftduring the surgical procedure.

It is usually preferred to administer a compound of formula I in theform of an acid addition salt, as is customary in the administration ofpharmaceuticals bearing a basic group, such as the piperidino ring. Itis also advantageous to administer such a compound by the oral route toan aging human (e.g. a post-menopausal female). For such purposes thefollowing oral dosage forms are available.

FORMULATIONS

In the formulations which follow, "Active ingredient" means a compoundof formula I or II.

Formulation 1: Gelatin Capsules

Hard gelatin capsules are prepared using the following:

    ______________________________________                                        Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Active ingredient 0.1-1000                                                    Starch, NF        0-650                                                       Starch flowable powder                                                                          0-650                                                       Silicone fluid 350 centistokes                                                                  0-15                                                        ______________________________________                                    

The ingredients are blended, passed through a No. 45 mesh U.S. sieve,and filled into hard gelatin capsules.

Examples of specific capsule formulations of the compound of formula 1wherein the compound is raloxifene, include those shown below:

Formulation 2: Raloxifene capsule

    ______________________________________                                        Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Raloxifene        1                                                           Starch, NF        112                                                         Starch flowable powder                                                                          225.3                                                       Silicone fluid 350 centistokes                                                                  1.7                                                         ______________________________________                                    

Formulation 3: Raloxifene capsule

    ______________________________________                                        Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Raloxifene        5                                                           Starch, NF        108                                                         Starch flowable powder                                                                          225.3                                                       Silicone fluid 350 centistokes                                                                  1.7                                                         ______________________________________                                    

Formulation 4: Raloxifene capsule

    ______________________________________                                        Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Raloxifene        10                                                          Starch, NF        103                                                         Starch flowable powder                                                                          225.3                                                       Silicone fluid 350 centistokes                                                                  1.7                                                         ______________________________________                                    

Formulation 5: Raloxifene capsule

    ______________________________________                                        Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Raloxifene        50                                                          Starch, NF        150                                                         Starch flowable powder                                                                          397                                                         Silicone fluid 350 centistokes                                                                  3.0                                                         ______________________________________                                    

The specific formulations above may be changed in compliance with thereasonable variations provided.

A tablet formulation is prepared using the ingredients below:

Formulation 6: Tablets

    ______________________________________                                        Ingredient       Quantity (mg/tablet)                                         ______________________________________                                        Active ingredient                                                                              0.1-1000                                                     Cellulose, microcrystalline                                                                    0-650                                                        Silicon dioxide, fumed                                                                         0-650                                                        Stearate acid    0-15                                                         ______________________________________                                    

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 0.1-1000 mg of active ingredientare made up as follows:

Formulation 7: Tablets

    ______________________________________                                        Ingredient          Quantity (mg/tablet)                                      ______________________________________                                        Active ingredient   0.1-1000                                                  Starch              45                                                        Cellulose, microcrystalline                                                                       35                                                        Polyvinylpyrrolidone                                                                              4                                                         (as 10% solution in water)                                                    Sodium carboxymethyl cellulose                                                                    4.5                                                       Magnesium stearate  0.5                                                       Talc                1                                                         ______________________________________                                    

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 60 U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yieldtablets.

Suspensions each containing 0.1-1000 mg of medicament per 5 mL dose aremade as follows:

Formulation 8: Suspensions

    ______________________________________                                        Ingredient           Quantity (mg/5 ml)                                       ______________________________________                                        Active ingredient    0.1-1000 mg                                              Sodium carboxymethyl cellulose                                                                     50 mg                                                    Syrup                1.25 mg                                                  Benzoic acid solution                                                                              0.10 mL                                                  Flavor               q.v.                                                     Color                q.v.                                                     Purified water to    5 mL                                                     ______________________________________                                    

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl cellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor, and color are diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

TEST PROCEDURE

Compounds of the invention have capacity to inhibit vascular smooth cellproliferation. This can be demonstrated by using cultured smooth cellsderived from rabbit aorta, proliferation being determined by themeasurement of DNA synthesis. Cells are obtained by explant method asdescribed in Ross, J. of Cell Biol 50:172 (1971). Cells are plated in 96well microtiter plates for five days. The cultures become confluent andgrowth arrested. The cells are then transferred to Dulbecco's ModifiedEagle's Medium (DMEM) containing 0.5-2% platelet poor plasma, 2 mML-glutamine, 100 U/ml penicillin, 100 μg ml streptomycin, 1 BC/ml ³H-thymidine, 20 ng/ml platelet-derived growth factor and varyingconcentrations of the compounds. Stock solution of compounds is preparedin dimethyl sulphoxide and then diluted to appropriate concentration(0.01-30 μM) in the above assay medium, Cells are then incubated at 37°C. for 24 hours under 5% CO₂ /95% air. At the end of 24 hours, the cellsare fixed in methanol. 3H thymidine incorporation in DNA was thendetermined by scintillation counting as described in Bonin et al., Exp.Cell Res. 181: 475-482 (1989).

Inhibition of smooth muscle cell proliferation by the compounds of theinvention is further demonstrated by determining their effects onexponentially growing cells. Smooth muscle cells from rabbit aortae areseeded in 12 well tissue culture plates in DMEM containing 10% fetalbovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/mlstreptomycin. After 24 hours, the cells are attached, the medium isreplaced with DMEM containing 10% serum, 2 mM L-glutamine, 100 U/mlpenicillin, 100 μg/ml streptomycin and indicated concentrations of thecompounds. Cells are allowed to grow for four days. Cells are treatedwith trypsin and number of cells in each cultures is determined bycounting using a ZM-Coulter counter.

Activity in the above tests indicates that the compounds of theinvention are of potential in the treatment of restenosis.

We claim:
 1. A method of inhibiting vascular smooth muscle cell proliferation comprising administering to a human or other mammal in need of treatment an effective amount of a compound having the formula ##STR8## ##STR9## wherein Ar is optionally substituted phenyl; ##STR10## R₄ is hydrogen or --OR₁, and pharmaceutically acceptable salts and solvates thereof.
 2. The method of claim 1 wherein said compound is the hydrochloride salt thereof.
 3. The method of claim 1 wherein said administration is prophylactic.
 4. A method of inhibiting vascular restinosis comprising administering to a human or other mammal in need of treatment an effective amount of a compound having the formula ##STR11## ##STR12## wherein Ar is optionally substituted phenyl; R₂ is ##STR13## R₄ is hydrogen or --OR₁, and pharmaceutically acceptable salts and solvates thereof.
 5. The method of claim 4 wherein said compound is the hydrochloride salt thereof.
 6. The method of claim 4 wherein said administration is prophylactic. 